Effective method of treatment of porcine circovirus and lawsonia intracellularis infections

ABSTRACT

The present invention relates to the use of porcine circovirus type 2 (PCV2) antigen and  Lawsonia intracellularis  ( L. intracellularis ) antigen for the prevention, reduction in severity of, lessening of the clinical symptoms associated therewith, reduced incidence in a herd, and treatment of swine against Porcine Circovirus Associated Diseases (PCVAD) and  L. intracellularis  associated diseases. In particular, the present invention provides a method for the treatment or prophylaxis of an animal against PCV2 infection and ileitis caused by  L. intracellularis , or a method for reduction of clinical symptoms caused by or associated with a PCV2 and  L. intracellularis  infection, comprising the step of administering an effective amount of PCV2 antigen and  L. intracellularis  antigen to an animal in need of such treatment.

RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 60/869,512. The teachings and content of thatapplication are hereby incorporated by reference herein.

SEQUENCE LISTING

A sequence listing in electronic format is being provided herewith.Applicants note that the sequence listing provided herewith is identicalto the sequence listing of WO06/072065, which is entirely incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of porcine circovirus type 2(PCV2) antigen and Lawsonia intracellularis (L. intracellularis) antigenfor the prevention, reduction of clinical symptoms associated with, andtreatment of swine against Porcine Circovirus Associated Diseases(PCVAD) and L. intracellularis associated diseases.

2. Background of the Invention

Porcine circovirus type 2 (PCV2) is a small (17-22 nm in diameter),icoshedral, non-enveloped DNA virus, which contains a single-strandedcircular genome. PCV2 shares approximately 80% sequence identity withporcine circovirus type 1 (PCV1). However, in contrast with PCV1, whichis generally non-virulent, infection of swine with PCV2 has recentlyassociated with a number of disease syndromes which have beencollectively named Porcine Circovirus Associated Diseases (PCVAD) (Allanet al, 2006, IPVS Congress). Postweaning Multisystemic Wasting Syndrome(PMWS) is generally regarded to be the major clinical manifestation ofPCVAD. (Harding et al., 1997, Swine Health Prod; 5: 201-203; Kennedy etal., 2000, J Comp Pathol; 122: 9-24). PMWS affects pigs between 5-18weeks of age. PMWS is clinically characterized by wasting, paleness ofthe skin, unthriftiness, respiratory distress, diarrhea, icterus, andjaundice. In some affected swine, a combination of all symptoms will beapparent while other affected swine will only have one or two of thesesymptoms. (Muirhead, 2002, Vet. Rec.; 150: 456) During necropsy,microscopic and macroscopic lesions also appear on multiple tissues andorgans, with lymphoid organs being the most common site for lesions.Allan and Ellis, 2000; J Vet. Diagn. Invest., 12: 3-14). A strongcorrelation has been observed between the amount of PCV2 nucleic acid orantigen and the severity of microscopic lymphoid lesions. Mortalityrates for swine infected with PCV2 can approach 80%. In addition toPMWS, PCV2 has been associated with several other infections includingpseudorabies, porcine reproductive and respiratory syndrome (PRRS),Glasser's disease, streptococcal meningitis, salmonellosis, postweaningcolibacillosis, dietetic hepatosis, and suppurative bronchopneumonia.However, research thus far has not confirmed whether any of theseclinical symptoms are in fact, the direct result of a PCV2 infection.Moreover, it is not yet known whether any of these clinical symptoms canbe effectively reduced or cured by an active agent directed againstPCV2.

Approaches to treat PCV2 infections based on a DNA vaccine are describedin U.S. Pat. No. 6,703,023. In WO 03/049703, production of a livechimeric vaccine is described. This vaccine comprises a PCV1 backbone inwhich an immunogenic gene of a pathogenic PCV2 strains replaces a geneof the PCV1 backbone. WO 99/18214 has provided several PCV2 strains andprocedures for the preparation of a killed PVC2 vaccine. An effectiveORF-2 based subunit vaccine has also been reported in WO 06/072065.

With growing prevalence of porcine circovirus type 2 associated disease(PCVAD) in nursery and finishing pigs in swine herds, accurate diagnosisof the disease is critical for effective health management within theswine industry. That is because some diseases, such as ileitis caused byLawsonia intracellularis can cause clinical symptoms—e.g. diarrhea andweight loss—in nursery or finishing stage of pigs that can be confusedwith porcine circovirus type 2 associated disease (PCVAD).

Ileitis or Proliferative enteritis (PE) caused by L. intracellularis isa disease of high economic impact in swine worldwide. L. intracellularisis an obligate, intracellular bacterium, described for example in S.McOrist et al., Infection and Immunity, Vol. 61, No. 19, 4286-4292(1993) and G. Lawson et al., J. of Clinical Microbiology, Vol. 31, No.5, 1136-1142 (1993). The disease was first identified in swine andcharacterized by its gross and microscopic pathology, and later by thedemonstration of the intracellular bacteria within affected cells. Thecharacterizing pathological feature of the disease is the proliferationof immature epithelial cells in the crypts of the ileum (terminal partof the small intestine), the large intestine or both. Sections ofinfected tissue are characterized by a reddened thickening mucosaresembling a “garden hose,” and enteric lesions. The gut thickeningultimately prevents normal gut function, absorption capabilities, andnutrient transfer. Clinical effects of the disease are chronic weightloss, unthriftiness, diarrhea, and death. The disease is of economicimportance owing to death loss, increased medication costs, poor weightgain and decreased food conversion in affected animals. Clinical casesof ileitis are observed most notably in pigs 6-20 weeks of age. However,the presence of L. intracellularis has been confirmed by polymerasechain reaction (PCR) in recently weaned pigs (3-4 weeks of age),suggesting subclinical L. intracellularis exposure occur in the nurseryand perhaps, originates from Lawsonia-positive dams (Mauch and Bilkei(2004) Vet Rec 155: 532; Marsteller et al. (2003). Swine Health Prod11:127-130; Stege et al. (2004) Vet Micro 104: 197-206). Currentvaccination strategies for the prevention treatment of proliferativeenteritis are limited to swine. For example, U.S. Pat. Nos. 5,714,375and 5,885,823 as well as WO 05/011731, all of which are hereinincorporated by reference in their entireties, provide vaccines for theimmunization of swine. Those vaccines are highly effective and known inthe market as Enterisol® Ileitis or Enterisol® Ileitis B3903 (BoehringerIngelheim Vetmedica Inc., St Joseph, Mo., USA).

DISCLOSURE OF THE INVENTION

Even if PCVAD, caused by PCV2, may cause similar clinical symptoms asileitis in swine and also reduce productivity of swine industry, itseffects were yet not considered to serve or prolong ileitis. However,due to the confusion in clinical signs caused by both pathogens, PCV2and L. intracellularis, a risk exists in respect to the vaccinationstrategy for swine herds and swine farms. Yet, no kind of co-vaccinationstrategy exists for the prevention or treatment of pigs against PCVADand ileitis caused by L. intracellularis, in particular due to theabsence of the specific awareness of that problem.

The present invention has identified that problem, and overcomes it byproviding a distinct advance in the state of the art. According to ageneral aspect, the present invention provides a method for thetreatment or prophylaxis of an animal against a PCV2 infection andileitis caused by L. intracellularis, or a method for reduction ofclinical symptoms caused by or associated with a PCV2 and L.intracellularis infection, comprising the step of administering aneffective amount of PCV2 antigen and L. intracellularis antigen to ananimal in need of such treatment. Preferably, said animal is swine. Thetreatment of swine with an appropriate PCV2 antigen and L.intracellularis antigen results in a better performance of thevaccinated pigs, in particular it results in a higher resistance againstPCVAD and ileitis. Moreover, the immunogenic potential of the PCV2antigen, preferably of Ingelvac® CircoFLEX™ (Boehringer IngelheimVetmedica, Inc, St Joseph, Mo., USA) can be enhanced by theadministration of L. intracellularis antigen, preferably by Enterisol®Ileitis or Enterisol® Ileitis B3903 (Boehringer Ingelheim VetmedicaInc., St Joseph, Mo., USA) and vice versa. PCV2 clinical signs anddisease manifestations are greatly magnified when L. intracellularisinfection is present and vice versa.

The immunogenic compositions and vaccination strategies as providedherewith can reduce and lessen these effects greatly, and more thanexpected. In other words, an unexpected synergistic effect can beachieved when animals, preferably pigs, are treated with PCV2 antigenand L. intracellularis antigen. Preferably the treatment with PCV2 andL. intracellularis antigen occurs prior to any PCV2 and/or L.intracellularis infection of swine.

The term “prevention” or “treatment” as used herein means, but is notlimited to a process which includes the administration of an L.intracellularis and PCV2 antigen to an animal, wherein said L.intracellularis antigen, when administered to said animal elicits or isable to elicit an immune response in said animal against L.intracellularis and wherein said PCV2 antigen, when administered to saidanimal elicits or is able to elicit an immune response in said animalagainst PCV2. Altogether, such treatment results in reduction of theclinical symptoms of PCVAD and/or ileitis.

The term reduction of clinical symptoms shall mean, but not limited tothe reduction of any of the clinical symptoms associated with a L.intracellularis and/or PCV2 infection. For example, it should refer toany reduction of pathogen load, pathogen shedding, reduction intransmission of and reduction loss of weight gain and the extentdiarrhea.

PCV2 Antigens

The term “PCV2 antigen”, as used herein, refers to any immunogeniccomposition which can be used to prevent or treat a PCV2infection-associated disease or condition in a subject. The term“antigen”, as used herein, refers in general to an amino acid sequence,or nucleic acid, which elicits an immune response in a subject, whenadministered to that subject. Thus, an antigen can be of proteineous ornucleic acid origin. A proteineous antigen, as used herein, includes thefull-length sequence of any immunogenic protein, as well as analogs orfragments thereof. In contrast, a nucleic acid antigen, as used herein,refers to a nucleic acid molecule that codes for a full-length sequenceof any immunogenic protein, as well as analogs or fragments thereof. Theterm “immunogenic protein, analog or fragment” refers to a fragment of aprotein which includes one or more epitopes and thus elicits the immuneresponse in a host.

An “immune response” means but is not limited to the development in asubset of a cellular and/or antibody-mediated immune response to thecomposition or vaccine of interest. Usually, an “immune response”includes but is not limited to one or more of the following effects: theproduction or activation of antibodies, B cells, helper T cells,suppressor T cells, and/or cytotoxic T cells, directed specifically toan antigen or antigens included in the composition or vaccine ofinterest. Preferably, the host will display either a therapeutic or aprotective immunological (memory) response such that resistance to newinfection will be enhanced and/or the clinical severity of the diseasereduced. Such protection will be demonstrated by either a reduction innumber or severity of, or lack of one or more of the symptoms associatedwith the infection of the pathogen, in the delay of onset of viremia, ina reduced viral persistence, in a reduction of the overall viral loadand/or in a reduction of viral excretion.

Thus a preferred PCV2 antigen, as used herein, can induce, stimulate orenhance the immune response against PCV2. In this context, the term PCV2antigen encompasses subunit immunogenic compositions, as well ascompositions containing whole killed, or attenuated and/or inactivatedPCV2.

By the term “subunit immunogenic composition”, a composition is meantthat contains at least one immunogenic polypeptide or antigen, but notall antigens, derived from or homologous to an antigen of interest. Sucha composition is substantially free of intact pathogen, e.g. PCV2. Thus,a “subunit immunogenic composition” is prepared from at least partiallypurified or fractionated (preferably substantially purified) immunogenicpolypeptides from PCV2, or recombinant analogs thereof. A preferredsubunit immunogenic composition comprises the PCV2 ORF-2 protein.

PCV2 ORF-2 protein is a highly conserved domain within PCV2 isolates andthereby, any PCV2 ORF-2 protein or DNA would be effective as the sourceof the PCV ORF-2 antigen. Preferred PCV2 ORF-2 proteins are thosedescribed in WO06/072065, in particular the PCV2 ORF-2 protein that isencoded by SEQ ID NO: 11. A further preferred PCV ORF-2 polypeptide isprovided as SEQ ID NO: 5 in WO06/072065. However, it is understood bythose of skill in the art that any of these sequences could vary by asmuch as 6-30% in sequence homology provided that they still retain theantigenic characteristics that render it useful in immunogeniccompositions. The antigenic characteristics of the PCV2 ORF-2 antigencan be, for example, estimated by the challenge experiment as providedby Example 4 of WO06/072065. The antigenic characteristic of a modifiedPCV ORF-2 antigen is, for example, still retained, when the modifiedantigen confers at least 70%, preferably at least 80%, even morepreferably more preferably 90% of the protective immunity as compared tothe PCV2 ORF-2 protein, encoded by the polynucleotide sequence of SEQ IDNO: 3 or SEQ ID NO: 4 as provided in WO06/072065.

Beside a full-length PCV2 ORF-2 protein, immunogenic portions of PCV2ORF-2 protein can also be used as PCV2 antigen. The term “immunogenicportion”, as used herein, refers to truncated and/or substituted forms,or fragments of PCV2 ORF-2 protein and/or polynucleotide, respectively.Preferably, such truncated and/or substituted forms, or fragments willcomprise at least 6 contiguous amino acids from the full-length PCV2ORF-2 protein. More preferably, the truncated or substituted forms, orfragments will have at least 10, more preferably at least 15, and stillmore preferably at least 19 contiguous amino acids from the full-lengthPCV2 ORF-2 protein. Two preferred sequences in this respect are providedas SEQ ID NO: 9 and SEQ ID NO: 10 of WO06/072065. It is furtherunderstood that such sequences may be a part of larger fragments ortruncated forms.

“Sequence Identity” as it is known in the art refers to a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol., 215:403-410 (1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 85%, preferably 90%, even more preferably 95% “sequenceidentity” to a reference nucleotide sequence, it is intended that thenucleotide sequence of the given polynucleotide is identical to thereference sequence except that the given polynucleotide sequence mayinclude up to 15, preferably up to 10, even more preferably up to 5point mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, in a polynucleotide having a nucleotidesequence having at least 85%, preferably 90%, even more preferably 95%identity relative to the reference nucleotide sequence, up to 15%,preferably 10%, even more preferably 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 15%, preferably 10%, evenmore preferably 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having a given aminoacid sequence having at least, for example, 85%, preferably 90%, evenmore preferably 95% sequence identity to a reference amino acidsequence, it is intended that the given amino acid sequence of thepolypeptide is identical to the reference sequence except that the givenpolypeptide sequence may include up to 15, preferably up to 10, evenmore preferably up to 5 amino acid alterations per each 100 amino acidsof the reference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 85%, preferably 90%, even morepreferably 95% sequence identity with a reference amino acid sequence,up to 15%, preferably up to 10%, even more preferably up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to15%, preferably up to 10%, even more preferably up to 5% of the totalnumber of amino acid residues in the reference sequence may be insertedinto the reference sequence. These alterations of the reference sequencemay occur at the amino or the carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in the one or more contiguous groups within thereference sequence. Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. However,conservative substitutions are not included as a match when determiningsequence identity.

“Sequence homology”, as used herein, refers to a method of determiningthe relatedness of two sequences. To determine sequence homology, two ormore sequences are optimally aligned, and gaps are introduced ifnecessary. However, in contrast to “sequence identity”, conservativeamino acid substitutions are counted as a match when determiningsequence homology. In other words, to obtain a polypeptide orpolynucleotide having 95% sequence homology with a reference sequence,85%, preferably 90%, even more preferably 95% of the amino acid residuesor nucleotides in the reference sequence must match or comprise aconservative substitution with another amino acid or nucleotide, or anumber of amino acids or nucleotides up to 15%, preferably up to 10%,even more preferably up to 5% of the total amino acid residues ornucleotides, not including conservative substitutions, in the referencesequence may be inserted into the reference sequence. Preferably thehomolog sequence comprises a stretch of at least 50, even morepreferably at least 100, even more preferably at least 250, and evenmore preferably at least 500 nucleotides.

A “conservative substitution” refers to the substitution of an aminoacid residue or nucleotide with another amino acid residue or nucleotidehaving similar characteristics or properties including size,hydrophobicity, etc., such that the overall functionality does notchange significantly.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide orpolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

Thus, the PCV2 antigen as used herein, can be a PCV2 antigen thatcomprises or consists of:

-   -   i) a PCV2 ORF-2 protein that comprises the sequence of SEQ ID        NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO:        11 of WO06/07065;    -   ii) a PCV2 ORF-2 protein that is at least 80% homologous to the        polypeptide of i),    -   iii) any immunogenic portion of the polypeptides of i) and/or        ii)    -   iv) the immunogenic portion of iii), comprising at least 10        contiguous amino acids included in the sequences of SEQ ID NO:        5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 of        WO06/072065,    -   v) a polypeptide that is encoded by a DNA comprising the        sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of WO06/072065.    -   vi) any polypeptide that is encoded by a polynucleotide that is        at least 80% homologous to the polynucleotide of v),    -   vii) any immunogenic portion of the polypeptides encoded by the        polynucleotide of v) and/or vi)    -   viii) the immunogenic portion of vii), wherein polynucleotide        coding for said immunogenic portion comprises at least 30        contiguous nucleotides included in the sequences of SEQ ID NO:        3, or SEQ ID NO: 4 of WO06/072065.

Preferably any of the immunogenic portions of PCV2 ORF-2 as describedabove have the immunogenic characteristics of PCV2 ORF-2 protein that isencoded by the sequence of SEQ ID NO: 3 or SEQ ID NO: 4 of WO 06/07065.

The PCV2 antigen, preferably any of the PCV2 ORF-2 proteins describedabove and as used in the immunogenic composition in accordance with thepresent invention can be derived in any fashion including isolation andpurification of PCV2 antigen, standard protein synthesis, andrecombinant methodology. Preferred methods for obtaining PCV2 ORF-2proteins are provided in WO06/072065, the teachings and content of whichare hereby incorporated by reference in their entirety. Briefly,susceptible cells are infected with a recombinant viral vectorcontaining PCV2 ORF-2 DNA coding sequences, PCV2 ORF-2 polypeptide isexpressed by the recombinant virus, and the expressed PCV2 ORF-2polypeptide is recovered from the supernatant by filtration andinactivated by any conventional method, preferably using binaryethylenimine, which is then neutralized to stop the inactivationprocess.

The PCV2 antigen, as used herein, can be a part of a PCV2 immunogeniccomposition that comprises i) any of the PCV2 ORF-2 proteins describedabove and ii) at least a portion of the viral vector expressing saidPCV2 ORF-2 protein, preferably of a recombinant baculovirus. Moreover,said PCV2 immunogenic composition can comprise i) any of the PCV2 ORF-2proteins described above, ii) at least a portion of the viral vectorexpressing said PCV2 ORF-2 protein, preferably of a recombinantbaculovirus, and iii) a portion of the cell culture supernatant.

The PCV2 immunogenic composition, as used herein, also refers to acomposition that comprises i) any of the PCV2 ORF-2 proteins describedabove, ii) at least a portion of the viral vector expressing said PCV2ORF-2 protein, iii) a portion of the cell culture, iv) and inactivatingagent, preferably BEI, to inactivate the recombinant viral vector,wherein about 90% of the components i) to iii) have a size smaller than1 μm. Preferably, BEI is present in concentrations effective toinactivate the baculovirus.

The PCV2 immunogenic composition, as used herein, also refers to acomposition that comprises i) any of the PCV2 ORF-2 proteins describedabove, ii) at least a portion of the viral vector expressing said PCV2ORF-2 protein, iii) a portion of the cell culture, iv) an inactivatingagent, preferably BEI, to inactivate the recombinant viral vector, andv) a neutralization agent to stop the inactivation mediated by theinactivating agent, wherein about 90% of the components i) to iii) havea size smaller than 1 μm. Preferably, if the inactivating agent is BEI,said composition comprises sodium thiosulfate in equivalent amounts toBEI.

Additionally, the PCV2 immunogenic composition, as used herein, caninclude one or more veterinary-acceptable carriers. As used herein, “aveterinary-acceptable carrier” includes any and all solvents, dispersionmedia, coatings, adjuvants, stabilizing agents, diluents, preservatives,antibacterial and antifungal agents, isotonic agents, adsorptiondelaying agents, and the like. Preferably, the PCV2 immunogeniccomposition comprises PCV2 ORF-2 protein, an adjuvant, preferablyCarbopol, and physiological saline. Diluents include water, saline,dextrose, ethanol, glycerol, and the like. Isotonic agents can includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin and alkali salts ofethylendiamintetracetic acid, among others. Adjuvants include aluminiumhydroxide and aluminium phosphate, saponins e.g., Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion,oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsions canbe based in particular on light liquid paraffin oil (EuropeanPharmacopea type); isoprenoid oil such as squalane or squalene oilresulting from the oligomerization of alkenes, in particular ofisobutene or decene; esters of acids or of alcohols containing a linearalkyl group, more particularly plant oils, ethyl oleate, propyleneglycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) orpropylene glycol dioleate; esters of branched fatty acids or alcohols,in particular isostearic acid esters. The oil is used in combinationwith emulsifiers to form the emulsion. The emulsifiers are preferablynonionic surfactants, in particular esters of sorbitan, of mannide (e.g.anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycoland of oleic, isostearic, ricinoleic or hydroxystearic acid, which areoptionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymerblocks, in particular the Pluronic products, especially L121. See Hunteret al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) andTodd et al., Vaccine 15:564-570 (1997). A further instance of anadjuvant is a compound chosen from the polymers of acrylic ormethacrylic acid and the copolymers of maleic anhydride and alkenylderivative. Advantageous adjuvant compounds are the polymers of acrylicor methacrylic acid which are cross-linked, especially with polyalkenylethers of sugars or polyalcohols. These compounds are known by the termcarbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in theart can also refer to U.S. Pat. No. 2,909,462 which describes suchacrylic polymers cross-linked with a polyhydroxylated compound having atleast 3 hydroxyl groups, preferably not more than 8, the hydrogen atomsof at least three hydroxyls being replaced by unsaturated aliphaticradicals having at least 2 carbon atoms. The preferred radicals arethose containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and otherethylenically unsaturated groups. The unsaturated radicals maythemselves contain other substituents, such as methyl. The products soldunder the name Carbopol (BF Goodrich, Ohio, USA) are particularlyappropriate. They are cross-linked with an allyl sucrose or with allylpentaerythritol. Among them, there may be mentioned Carbopol 974P, 934Pand 971P. Most preferred is the use of Carbopol, in particular the useof Carbopol 971P, preferably in amounts of about 500 μg to about 5 mgper dose, even more preferred in an amount of about 750 μg to about 2.5mg per dose and most preferred in an amount of about 1 mg per dose.Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314, or muramyl dipeptide among manyothers.

The PCV2 immunogenic composition, as used herein, also refers to animmunogenic composition that comprises i) any of the PCV2 ORF-2 proteinsdescribed above, ii) at least a portion of the viral vector expressingsaid PCV2 ORF-2 protein, iii) a portion of the cell culture, iv) aninactivating agent to inactivate the recombinant viral vector preferablyBEI, and v) an neutralization agent to stop the inactivation mediated bythe inactivating agent, preferably sodium thiosulfate in equivalentamounts to BEI; and vi) a suitable adjuvant, preferably Carbopol 971;wherein about 90% of the components i) to iii) have a size smaller than1 μm. According to a further aspect, the PCV2 immunogenic composition,as used herein, further comprises a pharmaceutical acceptable salt,preferably a phosphate salt in physiologically acceptableconcentrations. Preferably, the pH of said immunogenic composition isadjusted to a physiological pH, meaning between about 6.5 and 7.5.

The PCV2 immunogenic composition, as used herein, also refers toIngelvac CircoFLEX®, (Boehringer Ingelheim Vetmedica Inc, St Joseph,Mo., USA), CircoVac® (Merial SAS, Lyon, France), CircoVent (IntervetInc., Millsboro, Del., USA), or Suvaxyn PCV2 One Dose® (Fort DodgeAnimal Health, Kansas City, Kans., USA). The most preferred PCV2antigen, as used herein, is Ingelvac CircoFLEX®, (Boehringer IngelheimVetmedica Inc, St Joseph, Mo., USA)

Lawsonia intracellularis Antigens

The term “L. intracellularis” as used herein means the intracellular,curved gram-negative bacteria described in detail by C. Gebhart et al.,Int'l. J. of Systemic Bacteriology, Vol. 43, No. 3, 533-538 (1993) andS. McOrist et al., Int'l. J. of Systemic Bacteriology, Vol. 45, No. 4,820-825 (1995), each of which is incorporated herein by reference intheir entireties, and includes but is not limited to the isolatesdescribed in WO 96/39629 and WO 05/011731. In particular, the term “L.intracellularis” also means, but is not limited to the isolatesdeposited under the Budapest Treaty with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 andassigned ATCC accession number PTA 4926 or ATCC accession number 55783.Both isolates are described in WO 96/39629 and WO 05/011731,respectively. The term “L. intracellularis” also means, but is notlimited to any other L. intracellularis bacteria strain, or isolate,preferably having the immunogenic properties of at least one of the L.intracellularis strains described in WO 96/39629 and WO 05/011731, inparticular having the immunogenic properties of at least one of theisolates deposited under the Budapest Treaty with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209and assigned ATCC accession numbers PTA 4926 or ATCC accession number55783.

A strain or isolate has the “immunogenic properties” of at least one ofthe L. intracellularis strains described in WO 96/39629 and WO05/011731, in particular, of the isolates deposited as ATCC accessionnumbers PTA 4926 or ATCC accession number 55783, when it is detectableat least with one of the anti-L. intracellularis specific antibodies,described in WO06/01294, in an detection assay that is also described inWO06/01294. Preferably those antibodies are selected from the antibodieshaving the reference numbers 301:39, 287:6, 268:29, 110:9, 113:2 and268:18. Preferably, the detection assay is a sandwich ELISA as describedin Examples 2 and 3 of WO06/12949, whereas antibody 110:9 is used as acapture antibody and antibody 268:29 is used as a conjugated antibody.All antibodies disclosed in WO 06/12949 are produced by hybridoma cells,which are deposited at the Centre for Applied Microbiology and Research(CAMR) and European Collection of Cell Cultures (“ECACC”), Salisbury,Wiltshire SP4 OJG, UK, as patent deposits according to the BudapestTreaty. The date of deposit was May 11, 2004. HYBRIDOMA CELL LINE 110:9is successfully deposited under ECACC Acc. No. 04092204. HYBRIDOMA CELLLINE 113:2 is successfully deposited under ECACC Acc. No. 04092201.HYBRIDOMA CELL LINE 268:18 is successfully deposited under ECACC Acc.No. 04092202. HYBRIDOMA CELL LINE 268:29 is successfully deposited underECACC Acc. No. 04092206. HYBRIDOMA CELL LINE 287:6 is successfullydeposited under ECACC Acc. No. 04092203. HYBRIDOMA CELL LINE 301:39 issuccessfully deposited under ECACC Acc. No. 04092205.

The term “L. intracellularis antigen” as used herein means, but is notlimited to any composition of matter, that comprises at least oneantigen that can induce, stimulate or enhance the immune responseagainst a L. intracellularis-caused infection, when administered to ananimal. Preferably, said L. intracellularis antigen is a complete L.intracellularis bacterium, in particular in an inactivated form (a socalled killed bacterium), a modified live or attenuated L.intracellularis bacterium (a so called MLB), any sub-unit, polypeptideor component of L. intracellularis, or any chimeric vector eachcomprises at least an immunogenic amino acid sequence of L.intracellularis. The terms “immunogenic protein”, “immunogenicpolypeptide” or “immunogenic amino acid sequence” as used herein referto any amino acid sequence which elicits an immune response in a hostagainst a pathogen comprising said immunogenic protein, immunogenicpolypeptide or immunogenic amino acid sequence. In particular, an“immunogenic protein”, “immunogenic polypeptide” or “immunogenic aminoacid sequence” of L. intracellularis means any amino acid sequence thatcodes for an antigen which elicits an immunological response against L.intracellularis in a host to which said “immunogenic protein”,“immunogenic polypeptide” or “immunogenic amino acid sequence” isadministered.

Suitable L. intracellularis antigens include, but are not limited tothose described in EP 1219711; U.S. Pat. No. 6,605,696; WO 96/39629;WO97/20050; WO 00/69903; WO 00/69904; WO 00/69905; WO 00/69906; WO02/38594; WO 02/26250; WO 03/06665; WO 04/033631; WO 05/026200; WO05/011731; WO 06/116763; and/or WO 06/113782, which are all incorporatedentirely herein by reference.

Preferably, the L. intracellularis antigen is a modified live L.intracellularis bacteria. Most preferably, said L. intracellularisantigen is Enterisol® Ileitis or Enterisol® Ileitis B3903 (BoehringerIngelheim Vetmedica, Inc.).

Effective Amount of Porcine Circovirus and L. intracellularis Antigen

The amount of antigen that is effective to elicit an immune response oris able to elicit an immune response in an animal depends on theingredients of the vaccine and the schedule of administration.

PCV2 Antigen:

Typically, when an inactivated virus or a modified live viruspreparation is used in the combination vaccine, an amount of the vaccinecontaining about 10^(2.0) to about 10^(9.0) TCID₅₀ per dose, preferablyabout 10^(3.0) to about 10^(8.0) TCID₅₀ per dose, more preferably, about10^(4.0) to about 10^(8.0) TCID₅₀ per dose. In particular, when modifiedlive PCV2 is used in the vaccines, the recommended dose to beadministered to the susceptible animal is preferably about 10^(3.0)TCID₅₀ (tissue culture infective dose 50% end point)/dose to about10^(6.0) TCID₅₀/dose and more preferably about 10^(4.0) TCID₅₀/dose toabout 10^(5.0) TCID₅₀/dose. In general, the quantity of antigen will bebetween 0.2 and 5000 micrograms, and between 10^(2.0) and 10^(9.0)TCID₅₀, preferably between 10^(3.0) and 10^(6.0) TCID₅₀, and morepreferably between 10^(4.0) and 10^(5.0) TCID₅₀, when purified antigenis used.

Sub-unit viral vaccines are normally administered with an antigeninclusion level of at least 0.2 μg antigen per dose, preferably withabout 0.2 to about 400 μg/dose, still more preferably with about 0.3 toabout 200 μg/dose, even more preferably with about 0.35 to about 100μg/dose, still more preferably with about 0.4 to about 50 μg/dose, stillmore preferably with about 0.45 to about 30 μg/dose, still morepreferably with about 0.6 to about 15 μg/dose, even more preferably withabout 0.75 to about 8 μg/dose, even more preferably with about 1.0 toabout 6 μg/dose, and still more preferably with about 1.3 to about 3.0μg/dose.

If PCV ORF-2 antigen is used, the PCV ORF-2 antigen inclusion level isat least 0.2 μg/PCV2 ORF-2 protein as described above per dose of thefinal antigenic composition (μg/dose), more preferably from about 0.2 toabout 400 μg/dose, still more preferably from about 0.3 to about 200μg/dose, even more preferably from about 0.35 to about 100 μg/dose,still more preferably from about 0.4 to about 50 μg/dose, still morepreferably from about 0.45 to about 30 μg/dose, still more preferablyfrom about 0.6 to about 15 μg/dose, even more preferably from about 0.75to about 8 μg/dose, even more preferably from about 1.0 to about 6μg/dose, still more preferably from about 1.3 to about 3.0 μg/dose, evenmore preferably from about 1.4 to about 2.5 μg/dose, even morepreferably from about 1.5 to about 2.0 μg/dose, and most preferablyabout 1.6 μg/dose.

L. intracellularis:

Typically, when killed L. intracellularis antigen is used in thevaccine, the vaccine contains an amount of about 10⁵ to about 10⁹ colonyforming units (CFU) of the L. intracellularis bacterium per dose,preferably, about 10⁶ to about 10⁸ (CFU) of the bacterium per dose.

In particular, when modified live L. intracellularis bacteria are usedin the vaccines, e.g. the bacteria isolates designated isolate B3903,ATCC accession No. PTA-4926 and designated isolate N34NP40wk, ATCCaccession No. 55783 (both described in WO 96/39629 and WO 05/011731),the recommended dose to be administered to the susceptible animal ispreferably about 4.5 log 10 TCID₅₀ (tissue culture infective dose 50%end point)/dose to about 9.0 log 10 TCID₅₀/dose and more preferablyabout 4.9 log 10 TCID₅₀/dose to about 6.9 log 10 TCID₅₀/dose. In apreferred embodiment, the titer of the vaccine is about 5.9 log 10TCID₅₀/dose as determined by Tissue Culture Infective Dose 50% endpointdilution assay (TCID₅₀). In general, the quantity of immunogen will bebetween 50 and 5000 micrograms, and between 4.5 log 10 and 9.0 log 10TCID₅₀, more preferably between 4.9 log 10 and 6.9 log 10 TCID₅₀, whenpurified bacteria are used.

Sub-unit vaccines, for example those described in WO 06/116763 or WO06/113782, are normally administered with an antigen inclusion level ofat least 2 μg antigen per dose, preferably with about 2 to about 500μg/dose, still more preferably with about 5 to about 400 μg/dose, evenmore preferably with about 8 to about 300 μg/dose, still more preferablywith about 10 to about 200 μg/dose, still more preferably with about 10to about 150 μg/dose, still more preferably with about 10 to about 100μg/dose, still more preferably with about 10 to about 75 μg/dose, stillmore preferably with about 10 to about 50 μg/dose, and still morepreferably with about 10 to about 20 μg/dose. Administration preferablyoccurs via parenteral route such as intra muscularly or subcutaneouslyfor example.

Administration of PCV2 and L. intracellularis Antigen Method ofTreatment

As already mentioned above, according to a general aspect, the presentinvention provides a method for the treatment or prophylaxis of swineagainst a PCV2 infection and ileitis caused by L. intracellularis, or amethod for reduction of clinical symptoms caused by or associated with aPCV2 and L. intracellularis infection, comprising the step ofadministering an effective amount of PCV2 antigen and L. intracellularisantigen to an animal in need of such treatment. Preferably, the PCVantigen is Ingelvac® CircoFLEX™, and the L. intracellularis antigen isEnterisol® Ileitis or Enterisol® Ileitis B3903. Vaccination with both,PCV2 and L. intracellularis antigen should be done prior to infection ofpigs with PCV2 and/or L. intracellularis and can occur simultaneously orconsecutively. For example, the positive clinical effects seen by theprophylactic use of PCV2 antigen, preferably Ingelvac® CircoFLEX™ can beenhanced by the administration of L. intracellularis antigen, preferablyby Enterisol® Ileitis or Enterisol® Ileitis B3903. Administration ofPCV2 antigen, preferably Ingelvac® CircoFLEX™ prior to administration ofL. intracellularis antigen, e.g. Enterisol® Ileitis or Enterisol®Ileitis B3903 is preferred. Reduction of clinical symptoms associatedwith a PCV2 infection, enhance the effect of the L. intracellularisantigen when administered to pigs. However, administration of L.intracellularis antigen, preferably Enterisol® Ileitis or Enterisol®Ileitis B3903 prior to PCV2 antigen, preferably Ingelvac® CircoFLEX™ isalso advantageous for the overall performance of the pigs and within themeaning of the present invention.

It has been found that L intracellularis antigen, in particularEnterisol® Ileitis or Enterisol® Ileitis B3903 can be effectivelyadministered to pigs at day one (1) of age or later. Preferably,administration is done before week 12 of age. Thus, this finding allowsvaccination of pigs prior to their exposure to L. intracellularis. Asalready mentioned, treatment of pigs with L. intracellularis antigenenhances the positive clinical effects mediated by the PCV2 antigen.PCV2 antigen, in particular Ingelvac® CircoFLEX™ is administered to pigsat week three (3) of age or later. Preferably, PCV2 antigen, inparticular, Ingelvac® CircoFLEX™ is administered not later than week 12of age, preferably not later than week eight (8) of age, and morepreferably not later than week six (6) of age, because clinical symptomsof PCVAD often strikes when pigs are 8 to 16 weeks of age. Thus,according to one aspect, the L. intracellularis antigen, preferablyEnterisol® Ileitis or Enterisol® Ileitis B3903, is administered at dayone (1) of age or later, preferably at days one (1) to 21 of age,whereas the PCV2 antigen, preferably Ingelvac® CircoFLEX™ isadministered at week three (3) of age to week 12 of age, preferably toweek six (6) of age. The antigens are preferably administered in amountsas described above. In cases where Ingelvac® CircoFLEX™ and Enterisol®Ileitis or Enterisol® Ileitis B3903 are used, administration of one (1)dose of each antigen is preferred.

According to a further aspect, the interval between the administrationof L. intracellularis antigen and PCV2 antigen should be at least 1 to20 days. However, administration on the same day is also possible.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 2 to 20 days. According to a further aspect of the invention theinterval between the administration of L. intracellularis antigen andPCV2 antigen should be about 3 to 20 days. According to a further aspectof the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 4 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 5 to 20 days. According to a further aspect of the invention theinterval between the administration of L. intracellularis antigen andPCV2 antigen should be about 6 to 20 days. According to a further aspectof the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 7 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 8 to 20 days. According to a further aspect of the invention theinterval between the administration of L. intracellularis antigen andPCV2 antigen should be about 9 to 20 days. According to a further aspectof the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 10 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 11 to 20 days. According to a further aspect of the invention theinterval between the administration of L. intracellularis antigen andPCV2 antigen should be about 12 to 20 days. According to a furtheraspect of the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 13 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 14 to 20 days. According to a further aspect of the invention theinterval between the administration of L. intracellularis antigen andPCV2 antigen should be about 15 to 20 days. According to a furtheraspect of the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 16 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beat least 17 to 20 days. According to a further aspect of the inventionthe interval between the administration of L. intracellularis antigenand PCV2 antigen should be about 18 to 20 days. According to a furtheraspect of the invention the interval between the administration of L.intracellularis antigen and PCV2 antigen should be about 19 to 20 days.According to a further aspect of the invention the interval between theadministration of L. intracellularis antigen and PCV2 antigen should beabout 20 days.

As already mentioned, combined vaccination of pigs with both antigens(L. intracellularis and PCV2) reduces the overall porcine circovirusload and virus persistence in the body, as well as the immunosuppressiveeffect of porcine circovirus and therefore enhances the overallperformance of vaccinated pigs. Moreover, administration of PCV2antigen, preferably Ingelvac® CircoFLEX™ surprisingly can enhance theresistance against other pathogens and also significantly enhance thepotency of the L. intracellularis antigen, in particular with respect toreduction in loss of weight gain. Thus, according to a further aspect ofthe present invention, the administration of PCV2 antigen, preferablyIngelvac® CircoFLEX™ to pigs occurs at week three (3) to week 12 of age,preferably at week three (3) to week eight (8) of age week, morepreferably at week three (3) to week (6) of age and prior to orsimultaneous with the administration of the L. intracellularis antigen,preferably Enterisol® Ileitis or Enterisol® Ileitis B3903. Preferably,vaccination with PCV2 antigen occurs prior to vaccination with L.intracelluaris antigen, e.g. about 1 to 20 days prior to vaccinationwith L. intracellularis antigen. Even more preferably, vaccination withPCV2 antigen occurs about 2 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 3 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 4 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 5 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 6 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 7 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 8 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 9 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 10 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 11 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 12 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 13 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 14 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 15 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 16 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 17 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 18 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 19 to 20 days prior to vaccination with L.intracellularis antigen. Even more preferably, vaccination with PCV2antigen occurs about 20 days prior to vaccination with L.intracellularis antigen.

According to a further aspect, at least one further dose of L.intracellularis and/or PCV2 antigen as described above is administeredto the pigs, wherein said subsequent administration(s) are given atleast 14 days beyond the initial or any former administration. The PCV2antigen and/or L. intracellularis antigen can be administered with animmune stimulant. However, in cases of Ingelvac® CircoFLEX™ andEnterisol® Ileitis or Enterisol® Ileitis B3903, an immune stimulant canbe used, but its use is not necessary. Preferably, said immune stimulantis given at least twice. Preferably, at least 3 days, more preferably atleast 5 days, and even more preferably at least 7 days are in betweenthe first and the second or any further administration of the immunestimulant. Preferably, the immune stimulant is given at least 10 days,preferably 15 days, even more preferably 20, and even more preferably atleast 22 days beyond the initial administration of the PCV2 and/or L.intracellularis antigens as described above. A preferred immunestimulant is, for example, keyhole limpet hemocyanin (KLH), preferablyemulsified with incomplete Freund's adjuvant (KLH/ICFA). However, it isherewith understood, that any other immune stimulant known to a personskilled in the art can also be used. The term “immune stimulant” as usedherein, means any agent or composition that can trigger the immuneresponse, preferably without initiating or increasing a specific immuneresponse, for example the immune response against a specific pathogen.It is further instructed to administer the immune stimulant in asuitable dose.

Preferably on the specific antigen administered, the L. intracellularisantigen and/or PCV2 antigen may be applied through intravenous,intravascular, intramuscular, intranasal, intraarterial,intraperitoneal, oral, subcutaneous, intradermal, intracutaneous,intralobal, intramedullar, or intrapulmonary routes. In case ofIngelvac® CircoFLEX™, intramuscular administrations are preferred. Incases of Enterisol® Ileitis or Enterisol® Ileitis B3903, oralapplication is preferred.

Besides a general vaccination of herds with L. intracellularis antigenand PCV2 antigen in order to reduce clinical symptoms caused by orassociated with PCV2 infection and/or L. intracellularis, an individualvaccination program can be developed for each farm or swine herd whichis affected by swine pathogens, preferably by PCV2 and L.intracellularis. As already mentioned, incidence of L. intracellularisand PCV2 within a swine herd can affect the efficacy of the treatmentwith PCV2 antigen and L. intracellularis treatment. In particular,incidence of PCV2 can affect efficacy of the treatment with L.intracellularis and vice versa. It is therefore appropriate to estimatefirst the incidence of L. intracellularis and/or PCV2 in a herd of afarm prior to vaccination with L. intracellularis antigen and/or PCV2antigen. Incidence of L. intracellularis for example can be estimated byany diagnostic assay, which allows the detection L. intracellularisantigen or specific anti-L. intracellularis antibodies. Specificembodiments of those assays are described for example in WO 06/0202730and WO 06/012949. PCV2 incidence can be estimated by an assay describedin WO06/072065. After incidence is estimated, an individual vaccinationprogram can be developed, which reflects the PCV2 and L. intracellularisincidence of that farm or herd. Thus, according to a further aspect thepresent invention relates to a method for the treatment or prophylaxisof an animal, preferably swine against a PCV2 infection and ileitiscaused by L. intracellularis, or a method for reduction of clinicalsymptoms caused by or associated with a PCV2 and/or L. intracellularisinfection, comprising the steps of:

-   -   i. detecting the incidence of L. intracellularis and/or PCV2 in        a swine herd,    -   ii. administering to pigs of L. intracellularis positive herds        an effective amount of L. intracellularis antigen and/or PCV2        antigen; and/or    -   iii. administering to pigs of PCV2 positive herds an effective        amount of L. intracellularis antigen and/or PCV2 antigen.

Preferably, administration of L. intracellularis antigen and/or PCV2antigen occurs prior to infection of pigs with L. intracellularis and/orPCV2, most preferably prior to exposure of pigs with L. intracellularis,and administration of PCV2 antigen should be done prior to infection ofpigs with PCV2. Suitable administration regimes are described above.

Kit of Parts

A further aspect relates to a kit, comprising a container whichcomprises any of the PCV2 antigens as described herein, preferablyIngelvac® CircoFLEX™, a container which comprises any of the L.intracellularis antigens as described herein, preferably Enterisol®Ileitis or Enterisol® Ileitis B3903, and an instruction manual,including the information for the administration of the PCV2 and L.intracellularis antigen. Preferably, the instruction manual includes theadministration information as described supra for the co-vaccination ofpigs with PCV2 and L. intracellularis antigen. For example saidinstruction manual comprises the information, that pigs should betreated with the PCV2 antigen and/or L. intracellularis antigen prior toinfection of said pigs with PCV2 and/or L. intracellularis. Moreover,said instruction manual also can comprise the information, that pigsshould be vaccinated with the L. intracellularis antigen prior to thevaccination with the PCV2 antigen. According to a further aspect, theinstruction manual comprises the information, that the PCV2 antigenshould be administered prior to the administration of L.intracellularis. The sequence in treatment may be influenced by theincidence of both pathogens within a herd or farm and the effects of oneantigen to the other. For example, L. intracellularis antigen enhancesthe immunological potency of the PCV2 antigen, whereas the reduction ofclinical symptoms caused by or associated with PCV2 can enhance theeffectiveness of the L. intracellularis antigen, in particular inrespect to reduction in loss of weight gain. Thus, in cases whereincidence of PCV is predominant, vaccination against PCV2 should beperformed first and vice versa. Moreover the instruction manual can alsocomprise the information, that the pigs can be treated with the L.intracellularis antigen at day 1 of age or later, but preferably notlater than week 12 of age. Moreover, the instruction manual can alsocomprise the information, that the vaccination with the PCV2 antigenshould occur at week three (3) of age or later, but preferably not laterthan at week 12 of age, preferably not later than at week (eight) 8,most preferably not later than at week six (6) of age. Moreover, theinstruction manual also comprises the information that it is moreeffective to vaccinate the pigs consecutively with an interval inbetween the administration of the two antigens of about one (1) to 20days, and more specifically as described above. In the case of Ingelvac®CircoFLEX™, the instruction manual also includes the information thateach pig is preferably treated with one (1) dose intramuscularly. In thecase of Enterisol® Ileitis or Enterisol® Ileitis B3903, the instructionmanual also comprises the information, that pigs are preferably treatedwith one (1) dose administered orally. Moreover, according to a furtheraspect, said instruction manual can comprise the information of a secondor further administration(s) of at least one further dose of a PCV2and/or L. intracellularis antigen, wherein any subsequent administrationis at least 14 days beyond the initial or any former administration.Preferably, said instruction manual also includes the information, toadminister an immune stimulant. Preferably, said immune stimulant shallbe given at least twice. Preferably, at least 3, more preferably atleast 5, even more preferably at least 7 days are between the first andthe second or any further administration of the immune stimulant.Preferably, the immune stimulant is given at least 10 days, preferably15, even more preferably 20, even more preferably at least 22 daysbeyond the initial administration of the PCV2 and/or L. intracellularisantigen. A preferred immune stimulant is for example is keyhole limpethemocyanin (KLH), still preferably emulsified with incomplete Freund'sadjuvant (KLH/ICFA). However, it is herewith understood, that any otherimmune stimulant known to a person skilled in the art can also be used.“Immune stimulant” as used herein, means any agent or composition thatcan trigger the immune response, preferably without initiating orincreasing a specific immune response, for example the immune responseagainst a specific pathogen. It is further instructed to administer theimmune stimulant in a suitable dose. Moreover, the kit may also comprisea container, including at least one dose of the immune stimulant,preferably one dose of KLH, or KLH/ICFA.

1. A method for the treatment or prophylaxis of an animal against PCV2infection and ileitis caused by L. intracellularis, or a method forreduction of clinical symptoms caused by or associated with a PCV2 andL. intracellularis infection, comprising the step of administering aneffective amount of PCV2 antigen and L. intracellularis antigen to ananimal in need of such treatment.
 2. The method according to claim 1,wherein said animal is swine.
 3. The method according to claim 1,wherein said PCV2 antigen is an inactivated killed PCV2, modified lifePCV2, or a subunit of PVC2.
 4. The method according to claim 1, whereinsaid PCV2 antigen is PCV2 ORF2.
 5. The method according to claim 1,wherein said PCV2 antigen is selected from the group consisting ofIngelvac® CircoFLEX™, CircoVac® CircoVent or Suvaxyn PCV2 One Dose®. 6.The method according to claim 5, wherein said PCV2 antigen is Ingelvac®CircoFLEX™.
 7. The method according to claim 1, wherein said L.intracellularis antigen is a modified live L. intracellularis.
 8. Themethod according to claim 1, wherein said L. intracellularis antigen isEnterisol® Ileitis or Enterisol® Ileitis B3903.
 9. The method accordingto claim 1, wherein said PCV2 antigen is Ingelvac® CircoFLEX™ and the L.intracellularis antigen is Enterisol® Ileitis or Enterisol® IleitisB3903®.
 10. The method according to claim 1, wherein said L.intracellularis antigen is administered prior to the administration ofsaid PCV2 antigen.
 11. The method according to claim 1, wherein said L.intracellularis antigen is administered at day one of age to week 12 ofage.
 12. The method according to claim 1, wherein said PCV2 antigen isadministered prior to the administration of said L. intracellularisantigen.
 13. The method according to claim 1, wherein said PCV2 antigenis administered at week 3 to week 12 of age.
 14. The method according toclaim 1, wherein 2 to 20 days are in between the administration of bothantigens.
 15. The method according to claim 1, further comprising thestep of administering an immune stimulant.
 16. The method of claim 1,wherein prior to the administration of one or both antigens, theincidence of L. intracellularis and/or PCV2 is determined within a herdor farm.
 17. A kit of parts, comprising a container which comprises aPCV2 antigen, preferably Ingelvac® CircoFLEX™, a container whichcomprises L. intracellularis antigen, preferably Enterisol® Ileitis orEnterisol® Ileitis B3903, and an instruction manual, including theinformation for the administration of said PCV2 and L. intracellularisantigen.
 18. The kit of parts according to claim 17, wherein saidcontainer which comprises the PCV2 antigen and said container whichcomprises the L. intracellularis antigen are different containers. 19.The kit of parts according to claim 17, wherein said instruction manualincludes the information that the L. intracellularis antigen isadministered prior to the administration of the PCV2 antigen.
 20. Thekit of parts according to claim 17, wherein said instruction manualincludes the information that the L. intracellularis antigen isadministered at day one of age to week 12 of age.
 21. The kit of partsaccording to claim 17, wherein said instruction manual includes theinformation that the PCV2 antigen is administered prior to theadministration of the L. intracellularis antigen.
 22. The kit of partsaccording to claim 17, wherein said instruction manual includes theinformation that 2 to 20 days are in between the administration of bothantigens.
 23. The kit of parts according to claim 17, wherein saidinstruction letter includes the information that the PCV antigen isadministered at week 3 of age to week 12 of age.
 24. The kit of partsaccording to claim 17, further comprising an immune stimulant andwherein said instruction manual includes the information that an immunestimulant is also administered.
 25. The kit of parts according to claim17, wherein said instruction manual includes the information that theincidence of the L. intracellularis and/or PCV2 is determined within thefarm or specific herd prior to administration of the antigens.